EP0607064B1 - Testverfahren und Reagenz für Magnesium in menschlichen Körperflüssigkeiten - Google Patents

Testverfahren und Reagenz für Magnesium in menschlichen Körperflüssigkeiten Download PDF

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Publication number
EP0607064B1
EP0607064B1 EP19940400026 EP94400026A EP0607064B1 EP 0607064 B1 EP0607064 B1 EP 0607064B1 EP 19940400026 EP19940400026 EP 19940400026 EP 94400026 A EP94400026 A EP 94400026A EP 0607064 B1 EP0607064 B1 EP 0607064B1
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EP
European Patent Office
Prior art keywords
assay reagent
isocitrate
assay
chelating agent
magnesium
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Lifetime
Application number
EP19940400026
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English (en)
French (fr)
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EP0607064A1 (de
Inventor
Yukari Kawakami
Tsuyoshi Fujita
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Oriental Yeast Co Ltd
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Oriental Yeast Co Ltd
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Publication date
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Publication of EP0607064A1 publication Critical patent/EP0607064A1/de
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/84—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving inorganic compounds or pH
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C12Q1/32—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving dehydrogenase

Definitions

  • Magnesium is commonly found in most animal and plant cells, and magnesium ion is indispensable for growth and the sustaining of life.
  • magnesium in the body fluctuates in a competitive manner with calcium, and since such fluctuation has been observed in cases of dysfunction of the central nervous system or the heart, or in cases of kidney failure, acute pancreatitis, etc., magnesium assays are becoming important objects for examination.
  • An assay method for magnesium according to the present invention makes possible a quick, accurate assay of magnesium in human blood serum, and thus provides a great contribution to clinical examinations.
  • Mg++ reacts strongly and terminates the reaction according to formula (I), and therefore an accurate calibration line has been unobtainable, and it has been impossible to know the exact Mg++ content.
  • the present invention was accomplished for the purpose of an accurate assay of the Mg ++ content in untreated or treated body fluids.
  • the present invention it is possible to make an accurate assay of the Mg ++ content in untreated or treated body fluids, by using two separate assay reagents 1 and 2, with assay reagent 1 containing no iCDH, but containing a Mg ++ chelating agent, and assay reagent 2 containing iCDH and NADP + , and using both successively at a predetermined interval.
  • the method according to the present invention wherein the assay reagent 1 which contains no ICDH but contains a Mg ++ chelating agent and the assay reagent 2 which contains iCDH and NADP + are used separately, is superior for the assaying of Mg ++ in body fluids, and the measured values of Mg ++ according to the present invention are extremely accurate.
  • the fluid to be assayed containing Mg ++ may be blood serum, urine, or the like, and the measurement is made with the undiluted original solution.
  • a combined assay reagent for assaying magnesium in human body fluids comprising:
  • the assay reagent 1 may be composed by dissolving a Mg ++ chelating agent, a Mn ++ chelating agent and potassium isocitrate in a Tris buffer solution.
  • the assay reagent 2 may be composed by dissolving iCDH, NADP + and potassium phosphate in water.
  • the reaction may be conducted under heated conditions.
  • the assay reagent 1 is added to the untreated or treated body fluid, which is heated to about 37°C, and the Mg ++ is chelated for about 5 minutes.
  • the standing time is the period during which almost all of the Mg ++ has been chelated, and since the content of Mg ++ differs depending on the sample, it is necessary to preestablish a standing time for each sample which will produce an accurate straight line during the following reaction corresponding to the amount of Mg ++ .
  • the assay reagent 2 is added thereto, and under heated conditions of about 37°C the increase in the amount of NADPH from 2 to 3 minutes after the addition is measured based on the increase in the absorbance at 340 nm, and when this value is applied to the reference line the Mg ++ content in the original body fluid may be accurately known.
  • the Mg ++ chelating agent to be used according to the present invention may be any of the following substances or their salts.
  • EDTA trans-1,2-cyclohexanediamine-N,N,N',N'-tetraacetic acid, N,N-di(hydroxyethyl)glycine, 1,3-diaminopropane-2-ol-N,N,N',N'-tetraacetic acid, diethylenetriamine-N,N,N',N'',N''-pentaacetic acid, ethylenediamine-N,N'-diacetic acid, ethylenediamine-N,N'-propionic acid, N-hydroxyethylethylenediamine-N,N',N'-triacetic acid, ethylenediamine-N,N,N',N'-tetrakis(methylenephosphonic acid), glycol ether diamine-N,N,N',N'-tetraacetic acid, hexam
  • the iCDH is activated by manganese ion, but it is possible to avoid the influence of the manganese ion by including in the reaction solvent a chelating agent, for example GEDTA, which selectively traps manganese ion without trapping magnesium ion.
  • a chelating agent for example GEDTA
  • Mg ++ is deactivated by the Mg ++ chelating agent which contains no iCDH, and the property whereby the activity of iCDH increases precisely depending on the remaining minute amount of Mg ++ is used for a specific, quick, highly sensitive assay of magnesium.
  • Serum was separated from healthy human blood, 240 ⁇ l of the assay reagent 1 shown in Example 1 were added to 5 ⁇ l of the separated serum, and the mixture was allowed to stand at 37°C for 5 minutes, after which 60 ⁇ l of the assay reagent 2 shown in Example 1 were added thereto and the mixture was allowed to stand at 37°C for 2 minutes, the increase in the amount of absorbance at 340 nm during 2-3 minutes was measured, and rate assay was made, whereby a value of 2.5 mg Mg ++ /100 ml serum was obtained.
  • Example 2 To 5 ⁇ l of healthy human urine were added 240 ⁇ l of the assay reagent 1 shown in Example 1, and the mixture was allowed to stand at 37°C for 5 minutes, after which 60 ⁇ l of the assay reagent 2 shown in Example 1 were added thereto, the mixture was allowed to stand at 37°C for 2 minutes, the increase in the amount of absorbance at 340 nm during 2-3 minutes was measured, and rate assay was made, whereby a value of 8.0 mg Mg ++ /100 ml urine was obtained.

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  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • Zoology (AREA)
  • General Health & Medical Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Microbiology (AREA)
  • Urology & Nephrology (AREA)
  • Analytical Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Hematology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Genetics & Genomics (AREA)
  • Inorganic Chemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Cell Biology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biophysics (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Claims (9)

  1. Assay-Verfahren für Magnesium in menschlichen Körperflüssigkeiten, gekennzeichnet durch
    die Zugabe eines Assay-Reagenz 1, welches keine Isocitratdehydrogenase, aber einen Mg++-Chelatbildner enthält, zu einer unbehandelten oder behandelten Körperflüssigkeit, Stehenlassen der Mischung für einen vorherbestimmten Zeitraum, um beinahe das gesamte des Mg++ an den Mg++-Chelatbildner zu binden, und anschließende Zugabe eines Assay-Reagenz 2, welches Isocitratdehydrogenase und NADP+ enthält, Umwandeln des NADP+ in NADPH mit dem verbleibenden Mg++ und Messen der Menge an Mg++ in der menschlichen Körperflüssigkeit bezogen auf die NADPH-Erhöhung.
  2. Assay-Verfahren für Magnesium in menschlichen Körperflüssigkeiten, gekennzeichnet durch
    das Messen der Erhöhung der Menge an NADPH gemäß Anspruch 1 durch die Erhöhung in der Absorption bei 340 nm.
  3. Kombiniertes Assay-Reagenz zum Bestimmen von Magnesium in menschlichen Körperflüssigkeiten enthaltend
    ein einen Mg++-Chelatbildner und Isocitrat enthaltendes Assay-Reagenz 1 und
    ein Isocitratdehydrogenase und NADP+ enthaltendes Assay-Reagenz 2.
  4. Kombiniertes Assay-Reagenz nach Anspruch 3, worin das Assay-Reagenz weiter einen Mn++-Chelatbildner enthält.
  5. Kombiniertes Assay-Reagenz nach einem der Ansprüche 3 und 4, worin das Assay-Reagenz weiter Kaliumphosphat enthält.
  6. Assay-Verfahren nach einem der Ansprüche 1 und 2, worin das Assay-Reagenz 1 weiter Isocitrat enthält.
  7. Assay-Verfahren nach Anspruch 6, worin das Isocitrat Kaliumisocitrat ist.
  8. Assay-Verfahren für Magnesium in menschlichen Körperflüssigkeiten nach Anspruch 1,
    gekennzeichnet durch
    die Zugabe eines Assay-Reagenz 1, welches keine Isocitratdehydrogenase, aber einen Mg++-Chelatbildner, einen Mn++-Chelatbildner und Kaliumisocitrat enthält, zu einer behandelten oder unbehandelten Körperflüssigkeit, Stehenlassen der Mischung über einen vorherbestimmten Zeitraum, um beinahe das gesamte Mg++ an den Mg++-Chelatbildner zu binden, und anschließende Zugabe eines Assay-Reagenz 2, welches Isocitratdehydrogenase, NADP+ und Kaliumphosphat enthält, Umwandlung des NADP+ in NADPH mit dem verbleibenden Mg++ und Messen der Menge an Mg++ in der menschlichen Körperflüssigkeit bezogen auf die Erhöhung in der Menge an NADPH.
  9. Kombiniertes Assay-Reagenz nach einem der Ansprüche 3 und 5, worin das Isocitrat Kaliumcitrat ist.
EP19940400026 1993-01-14 1994-01-05 Testverfahren und Reagenz für Magnesium in menschlichen Körperflüssigkeiten Expired - Lifetime EP0607064B1 (de)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
JP05020837A JP3133186B2 (ja) 1993-01-14 1993-01-14 ヒト体液中のマグネシウムの定量方法及び試薬
JP2083793 1993-01-14
JP20837/93 1993-01-14

Publications (2)

Publication Number Publication Date
EP0607064A1 EP0607064A1 (de) 1994-07-20
EP0607064B1 true EP0607064B1 (de) 2000-04-05

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EP19940400026 Expired - Lifetime EP0607064B1 (de) 1993-01-14 1994-01-05 Testverfahren und Reagenz für Magnesium in menschlichen Körperflüssigkeiten

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EP (1) EP0607064B1 (de)
JP (1) JP3133186B2 (de)
DE (1) DE69423796T2 (de)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100353608B1 (ko) * 2000-05-06 2002-09-27 주식회사 소일테크 분광광도계를 이용한 토양 치환성 마그네슘 정량방법

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4657854A (en) * 1983-05-05 1987-04-14 Ivan Endre Modrovich Assay systems based on magnesium-responsive enzymes
EP0207493B1 (de) * 1985-07-02 1990-08-16 Oriental Yeast Co., Ltd. Verfahren zur Beendigung der Isozitratdehydrogenase-Reaktion
JP2748134B2 (ja) * 1988-11-29 1998-05-06 オリエンタル酵母工業株式会社 マグネシウムの定量方法

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Publication number Publication date
JPH06209792A (ja) 1994-08-02
EP0607064A1 (de) 1994-07-20
JP3133186B2 (ja) 2001-02-05
DE69423796T2 (de) 2000-11-23
DE69423796D1 (de) 2000-05-11

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